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1.
Cell ; 184(12): 3109-3124.e22, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34004145

RESUMO

Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These "glycoRNAs" were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.


Assuntos
Membrana Celular/metabolismo , Polissacarídeos/metabolismo , RNA/metabolismo , Animais , Anticorpos/metabolismo , Sequência de Bases , Vias Biossintéticas , Linhagem Celular , Sobrevivência Celular , Humanos , Espectrometria de Massas , Ácido N-Acetilneuramínico/metabolismo , Poliadenilação , Polissacarídeos/química , RNA/química , RNA/genética , RNA não Traduzido/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Coloração e Rotulagem
2.
Cell ; 174(1): 218-230.e13, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29804836

RESUMO

Ribonucleoprotein enzymes require dynamic conformations of their RNA constituents for regulated catalysis. Human telomerase employs a non-coding RNA (hTR) with a bipartite arrangement of domains-a template-containing core and a distal three-way junction (CR4/5) that stimulates catalysis through unknown means. Here, we show that telomerase activity unexpectedly depends upon the holoenzyme protein TCAB1, which in turn controls conformation of CR4/5. Cells lacking TCAB1 exhibit a marked reduction in telomerase catalysis without affecting enzyme assembly. Instead, TCAB1 inactivation causes unfolding of CR4/5 helices that are required for catalysis and for association with the telomerase reverse-transcriptase (TERT). CR4/5 mutations derived from patients with telomere biology disorders provoke defects in catalysis and TERT binding similar to TCAB1 inactivation. These findings reveal a conformational "activity switch" in human telomerase RNA controlling catalysis and TERT engagement. The identification of two discrete catalytic states for telomerase suggests an intramolecular means for controlling telomerase in cancers and progenitor cells.


Assuntos
RNA não Traduzido/química , Telomerase/metabolismo , Biocatálise , Linhagem Celular , Células HeLa , Humanos , Chaperonas Moleculares , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/metabolismo , Telomerase/antagonistas & inibidores , Telomerase/química , Telomerase/genética , Telômero/metabolismo
3.
Cell ; 152(6): 1298-307, 2013 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-23498938

RESUMO

In biology as in real estate, location is a cardinal organizational principle that dictates the accessibility and flow of informational traffic. An essential question in nuclear organization is the nature of the address code--how objects are placed and later searched for and retrieved. Long noncoding RNAs (lncRNAs) have emerged as key components of the address code, allowing protein complexes, genes, and chromosomes to be trafficked to appropriate locations and subject to proper activation and deactivation. lncRNA-based mechanisms control cell fates during development, and their dysregulation underlies some human disorders caused by chromosomal deletions and translocations.


Assuntos
Núcleo Celular/química , Doença/genética , RNA Longo não Codificante/química , Animais , Núcleo Celular/genética , Regulação da Expressão Gênica , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
4.
Mol Cell ; 80(4): 557-559, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33217314

RESUMO

In this issue of Molecular Cell,Sun et al. (2020) identify ERK-mediated phosphorylation of the m6A methyltransferase complex as a regulatory mechanism for m6A and pluripotency and highlight the potential of this interaction as a target for cancer therapy.


Assuntos
Processamento de Proteína Pós-Traducional , Metilação
5.
RNA ; 30(8): 1025-1040, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38684317

RESUMO

RNA modifications have a substantial impact on tRNA function, with modifications in the anticodon loop contributing to translational fidelity and modifications in the tRNA core impacting structural stability. In bacteria, tRNA modifications are crucial for responding to stress and regulating the expression of virulence factors. Although tRNA modifications are well-characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa, remains limited. Here, we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in Escherichia coli, which enabled us to identify similar modifications in P. aeruginosa Our analysis supports a substantial degree of conservation between the two organisms, while also uncovering potential sites of tRNA modification in P. aeruginosa tRNAs that are not present in E. coli The mutational signature at one of these sites, position 46 of tRNAGln1(UUG) is dependent on the P. aeruginosa homolog of TapT, the enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) modification. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA-modifying enzymes, some of which play roles in determining virulence and pathogenicity.


Assuntos
Escherichia coli , Pseudomonas aeruginosa , RNA de Transferência , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Processamento Pós-Transcricional do RNA , Anticódon/genética , Anticódon/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/química , Conformação de Ácido Nucleico
6.
Nat Rev Genet ; 21(11): 651-670, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32764716

RESUMO

All organisms must safeguard the integrity of their DNA to avoid deleterious consequences of genome instability, which have been linked to human diseases such as autoimmune disorders, neurodegenerative diseases and cancer. Traditionally, genome maintenance has been viewed largely in terms of DNA-protein interactions. However, emerging evidence points to RNA as a key modulator of genome stability, with seemingly opposing roles in promoting chromosomal instability and protecting genome integrity. Unravelling the mechanistic and contextual basis of this duality will not only improve our understanding of the interfaces between RNA and the genome but will also provide important insights into how disrupted RNA metabolism contributes to disease origin, laying the foundation for targeted intervention.


Assuntos
Genoma Humano , Instabilidade Genômica , RNA/fisiologia , Adenosina/metabolismo , Animais , Reparo do DNA , Células Eucarióticas , Humanos , RNA Polimerase II/metabolismo , Processamento Pós-Transcricional do RNA , Retroelementos , Transcrição Gênica
7.
Hum Mol Genet ; 32(22): 3135-3145, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37561409

RESUMO

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant condition characterized by the development of cutaneous and uterine leiomyomas and risk for development of an aggressive form of papillary renal cell cancer. HLRCC is caused by germline inactivating pathogenic variants in the fumarate hydratase (FH) gene, which encodes the enzyme that catalyzes the interconversion of fumarate and L-malate. We utilized enzyme and protein mobility assays to evaluate the FH enzyme in a cohort of patients who showed clinical manifestations of HLRCC but were negative for known pathogenic FH gene variants. FH enzyme activity and protein levels were decreased by 50% or greater in three family members, despite normal FH mRNA expression levels as measured by quantitative PCR. Direct Nanopore RNA sequencing demonstrated 57 base pairs of retained intron sequence between exons 9 and 10 of polyadenylated FH mRNA in these patients, resulting in a truncated FH protein. Genomic sequencing revealed a heterozygous intronic alteration of the FH gene (chr1: 241498239 T/C) resulting in formation of a splice acceptor site near a polypyrimidine tract, and a uterine fibroid obtained from a patient showed loss of heterozygosity at this site. The same intronic FH variant was identified in an unrelated patient who also showed a clinical phenotype of HLRCC. These data demonstrate that careful clinical assessment as well as biochemical characterization of FH enzyme activity, protein expression, direct RNA sequencing, and genomic DNA sequencing of patient-derived cells can identify pathogenic variants outside of the protein coding regions of the FH gene.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Leiomiomatose , Neoplasias Cutâneas , Neoplasias Uterinas , Feminino , Humanos , Carcinoma de Células Renais/genética , Leiomiomatose/genética , Leiomiomatose/patologia , Fumarato Hidratase/genética , Fumarato Hidratase/análise , Neoplasias Renais/genética , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Mutação , RNA Mensageiro/genética
8.
Mol Cell ; 67(2): 228-238.e5, 2017 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-28625551

RESUMO

Circular RNAs (circRNAs) are single-stranded RNAs that are joined head to tail with largely unknown functions. Here we show that transfection of purified in vitro generated circRNA into mammalian cells led to potent induction of innate immunity genes and confers protection against viral infection. The nucleic acid sensor RIG-I is necessary to sense foreign circRNA, and RIG-I and foreign circRNA co-aggregate in cytoplasmic foci. CircRNA activation of innate immunity is independent of a 5' triphosphate, double-stranded RNA structure, or the primary sequence of the foreign circRNA. Instead, self-nonself discrimination depends on the intron that programs the circRNA. Use of a human intron to express a foreign circRNA sequence abrogates immune activation, and mature human circRNA is associated with diverse RNA binding proteins reflecting its endogenous splicing and biogenesis. These results reveal innate immune sensing of circRNA and highlight introns-the predominant output of mammalian transcription-as arbiters of self-nonself identity.


Assuntos
Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Tolerância Imunológica , Imunidade Inata , Íntrons , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/imunologia , RNA/genética , RNA/imunologia , Animais , Sequência de Bases , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/metabolismo , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Tolerância Imunológica/genética , Imunidade Inata/genética , Camundongos , Conformação de Ácido Nucleico , Ligação Proteica , Células RAW 264.7 , RNA/biossíntese , RNA/química , RNA Circular , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Imunológicos , Spliceossomos/imunologia , Spliceossomos/metabolismo , Transfecção
9.
Cell ; 139(1): 135-48, 2009 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19804759

RESUMO

We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/análise , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/genética , Meiose , Metáfase , Mitose , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Uridina/metabolismo
10.
Cell ; 139(1): 123-34, 2009 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19804758

RESUMO

RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1-interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci but does not downregulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Animais , Caenorhabditis elegans/genética , RNA Helicases DEAD-box/metabolismo , RNA Polimerase Dependente de RNA/metabolismo
11.
Nature ; 548(7667): 338-342, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28792938

RESUMO

N6-methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by 'writers', 'erasers' and 'readers' of this mark. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that the deletion of m6A 'writer' protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopaenic mouse adoptive transfer model, naive Mettl3-deficient T cells failed to undergo homeostatic expansion and remained in the naive state for up to 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding the STAT signalling inhibitory proteins SOCS1, SOCS3 and CISH were marked by m6A, exhibited slower mRNA decay and showed increased mRNAs and levels of protein expression in Mettl3-deficient naive T cells. This increased SOCS family activity consequently inhibited IL-7-mediated STAT5 activation and T cell homeostatic proliferation and differentiation. We also found that m6A has important roles for inducible degradation of Socs mRNAs in response to IL-7 signalling in order to reprogram naive T cells for proliferation and differentiation. Our study elucidates for the first time, to our knowledge, the in vivo biological role of m6A modification in T-cell-mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.


Assuntos
Adenosina/análogos & derivados , Homeostase , Interleucina-7/imunologia , RNA Mensageiro/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Linfócitos T/citologia , Adenosina/metabolismo , Transferência Adotiva , Animais , Diferenciação Celular , Proliferação de Células , Colite/prevenção & controle , Proteínas de Ligação a DNA/deficiência , Modelos Animais de Doenças , Feminino , Masculino , Metilação , Metiltransferases/deficiência , Camundongos , Estabilidade de RNA , RNA Mensageiro/química , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo
12.
Nature ; 519(7544): 486-90, 2015 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-25799993

RESUMO

Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.


Assuntos
Regulação da Expressão Gênica , Conformação de Ácido Nucleico , RNA/química , RNA/genética , Acilação , Adenosina/análogos & derivados , Animais , Sítios de Ligação , Sobrevivência Celular , Química Click , Biologia Computacional , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/genética , Genoma/genética , Camundongos , Modelos Moleculares , Biossíntese de Proteínas/genética , RNA/classificação , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Ribonucleico/genética , Ribossomos/metabolismo , Transcriptoma/genética
14.
Mol Cell ; 36(2): 231-44, 2009 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-19800275

RESUMO

Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the Dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that, in the germline, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage structures called P granules. WAGO-1 silences certain genes, transposons, pseudogenes, and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest additional regulatory functions for small RNAs.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Genoma/genética , Células Germinativas/metabolismo , RNA de Helmintos/metabolismo , RNA Interferente Pequeno/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans/química , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Análise de Sequência de RNA , Temperatura
16.
Mol Cell ; 31(1): 67-78, 2008 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-18571452

RESUMO

In metazoans, Piwi-related Argonaute proteins have been linked to germline maintenance, and to a class of germline-enriched small RNAs termed piRNAs. Here we show that an abundant class of 21 nucleotide small RNAs (21U-RNAs) are expressed in the C. elegans germline, interact with the C. elegans Piwi family member PRG-1, and depend on PRG-1 activity for their accumulation. The PRG-1 protein is expressed throughout development and localizes to nuage-like structures called P granules. Although 21U-RNA loci share a conserved upstream sequence motif, the mature 21U-RNAs are not conserved and, with few exceptions, fail to exhibit complementarity or evidence for direct regulation of other expressed sequences. Our findings demonstrate that 21U-RNAs are the piRNAs of C. elegans and link this class of small RNAs and their associated Piwi Argonaute to the maintenance of temperature-dependent fertility.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , RNA de Helmintos/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas , Sequência de Bases , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Fertilidade , Regulação da Expressão Gênica , Células Germinativas/citologia , Células Germinativas/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Complexo de Inativação Induzido por RNA , Sequências Reguladoras de Ácido Nucleico/genética
17.
J Am Chem Soc ; 137(5): 2107-15, 2015 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-25611135

RESUMO

N(6)-Methyladenosine (m(6)A) modification is hypothesized to control processes such as RNA degradation, localization, and splicing. However, the molecular mechanisms by which this occurs are unclear. Here, we measured structures of an RNA duplex containing m(6)A in the GGACU consensus, along with an unmodified RNA control, by 2D NMR. The data show that m(6)A-U pairing in the double-stranded context is accompanied by the methylamino group rotating from its energetically preferred syn geometry on the Watson-Crick face to the higher-energy anti conformation, positioning the methyl group in the major groove. Thermodynamic measurements of m(6)A in duplexes reveal that it is destabilizing by 0.5-1.7 kcal/mol. In contrast, we show that m(6)A in unpaired positions base stacks considerably more strongly than the unmodified base, adding substantial stabilization in single-stranded locations. Transcriptome-wide nuclease mapping of methylated RNA secondary structure from human cells reveals a structural transition at methylated adenosines, with a tendency to single-stranded structure adjacent to the modified base.


Assuntos
Adenosina/análogos & derivados , RNA/química , Adenina/química , Adenina/metabolismo , Adenosina/química , Adenosina/metabolismo , Pareamento de Bases , Linhagem Celular , Humanos , Metilação , Modelos Moleculares , RNA/metabolismo , Estabilidade de RNA , Ribonucleases/metabolismo , Termodinâmica
18.
Curr Opin Genet Dev ; 86: 102206, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38788488

RESUMO

The modification N6-methyladenosine (m6A) plays an important role in determining the functional output of gene expression programs. Throughout the transcriptome, the levels of m6A are tightly regulated by the opposing activities of methyltransferases and demethylases, as well as the interaction of modified transcripts with m6A-dependent RNA-binding proteins that modulate transcript stability, often referred to as writers, erasers, and readers. The enzymatic activities of both writers and erasers are tightly linked to the cellular metabolic environment, as these enzymatic reactions rely on metabolism intermediaries as cofactors. In this review, we highlight the examples of intersection between metabolism and m6A-dependent gene regulation and discuss the different contexts where this interaction plays important roles.


Assuntos
Adenosina , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genética , Regulação da Expressão Gênica , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genética
19.
bioRxiv ; 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38529508

RESUMO

RNA modifications have a substantial impact on tRNA function, with modifications in the anticodon loop contributing to translational fidelity and modifications in the tRNA core impacting structural stability. In bacteria, tRNA modifications are crucial for responding to stress and regulating the expression of virulence factors. Although tRNA modifications are well-characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa, remains limited. Here we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in E. coli, which enabled us to identify similar modifications in P. aeruginosa. Our analysis revealed a substantial degree of conservation between the two organisms, while also uncovering potential sites of tRNA modification in P. aeruginosa tRNAs that are not present in E. coli. The mutational signature at one of these sites, position 46 of tRNAGln1(UUG) is dependent on the P. aeruginosa homolog of TapT, the enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) modification. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA modifying enzymes, some of which play roles in determining virulence and pathogenicity.

20.
Chem Biol Interact ; 394: 110989, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38574836

RESUMO

Although few resistance mechanisms for histone deacetylase inhibitors (HDACis) have been described, we recently demonstrated that TMT1A (formerly METTL7A) and TMT1B (formerly METTL7B) can mediate resistance to HDACis with a thiol as the zinc-binding group by methylating and inactivating the drug. TMT1A and TMT1B are poorly characterized, and their normal physiological role has yet to be determined. As animal model systems are often used to determine the physiological function of proteins, we investigated whether the ability of these methyltransferases to methylate thiol-based HDACis is conserved across different species. We found that TMT1A was conserved across rats, mice, chickens, and zebrafish, displaying 85.7%, 84.8%, 60.7%, and 51.0% amino acid sequence identity, respectively, with human TMT1A. Because TMT1B was not found in the chicken or zebrafish, we focused our studies on the TMT1A homologs. HEK-293 cells were transfected to express mouse, rat, chicken, or zebrafish homologs of TMT1A and all conferred resistance to the thiol-based HDACIs NCH-51, KD-5170, and romidepsin compared to empty vector-transfected cells. Additionally, all homologs blunted the downstream effects of HDACi treatment such as increased p21 expression, increased acetylated histone H3, and cell cycle arrest. Increased levels of dimethylated romidepsin were also found in the culture medium of cells transfected to express any of the TMT1A homologs after a 24 h incubation with romidepsin compared to empty-vector transfected cells. Our results indicate that the ability of TMT1A to methylate molecules is conserved across species. Animal models may therefore be useful in elucidating the role of these enzymes in humans.


Assuntos
Galinhas , Inibidores de Histona Desacetilases , Metiltransferases , Peixe-Zebra , Animais , Humanos , Camundongos , Ratos , Sequência de Aminoácidos , Sequência Conservada , Depsipeptídeos/farmacologia , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Metilação , Metiltransferases/metabolismo , Metiltransferases/genética , Especificidade da Espécie , Compostos de Sulfidrila/metabolismo , Peixe-Zebra/metabolismo
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